Application of fibroblast BALB / c-3T3 overexpressing specific protein in preparation of hybridoma
By overexpressing specific membrane proteins on fibroblast BALB/c-3T3 and directly immunizing mice, the high cost and low efficiency problems under the protein + adjuvant immune mode in the prior art were solved, and efficient and easy acquisition was achieved Target antibodies reduce preparation costs and protein consumption.
Patent Information
- Application Number
- CN202411462614.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-19
- Publication Date
- 2025-01-14
AI Technical Summary
In the prior art, hybridoma cells are prepared by relying on the protein + adjuvant immune mode, which is costly and inefficient, and requires multiple immunization of mice to obtain target antibodies, which increases protein consumption and preparation costs.
Fibroblasts BALB/c-3T3 overexpressing specific proteins were used to overexpress membrane proteins through lentiviral transfection and other methods to directly immunize mice, avoid the use of adjuvants, simplify the preparation process, and obtain the target antibody through cell fusion.
It achieves efficient and easy acquisition of target antibodies, reduces preparation costs, reduces protein consumption, improves the positive rate, and does not require the purchase of commercially available proteins, and the overall cost can be reduced to half of the original cost.
Smart Images
Figure CN119307459A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of medicine and biology, and particularly relates to the use of BALB / c-3T3 fibroblasts overexpressing a specific protein in the preparation of hybridomas. Background Art
[0002] Hybridoma technology is currently an important technology for obtaining mouse monoclonal and polyclonal antibodies, laying the foundation for the development of antibody drugs. Its inventor, Nils Jeni, won the 1984 Nobel Prize in Physiology or Medicine for this. Although there are currently technologies such as phage display library technology and single B cell antibody technology that can also be used for monoclonal antibody preparation, hybridoma technology still occupies an important position in this field due to its simple operation, mature technology, low cost, and good naturalness, specificity and stability of the prepared monoclonal antibodies. The steps of hybridoma preparation of monoclonal antibodies are divided into multiple stages, including antigen preparation, animal immunization, fusion of spleen cells of immunized animals with myeloma cells to obtain hybridoma cells, screening hybridoma cells to obtain positive hybridoma cells that secrete target antibodies, multiple subcloning to obtain monoclonal hybridoma cell lines that stably secrete antibodies, expansion of positive monoclonal hybridoma cells, and sequencing to obtain monoclonal antibody sequences.
[0003] At present, most researchers mainly use the target immune adjuvant to immunize mice, and some researchers use cells that naturally express the target antigen as particulate antigens to immunize mice, and a very small number of researchers use recombinant transfer plasmids instead of proteins. Generally speaking, the preparation of hybridomas derived from mouse monoclonal antibodies requires the preparation of 3 to 5 mice with normal immune function, with BALB / c mice being the most common. Each mouse needs to be immunized more than 3 times. Before cell fusion, the mouse with the highest antibody titer needs to be boosted once. The amount of protein used for each immunization is at least 50ug / mouse, so the protein consumption is large. Depending on the type, source, preparation method, label, sequence position and length, the price of 100ug large-package commercial protein generally ranges from 3,000 to 30,000 yuan, and the comprehensive cost is generally at least RMB 30,000 or more. Therefore, the cost of using protein for immunization is high, and protein, as a soluble antigen, needs to rely on the combination of immune adjuvants to enhance immunogenicity (i.e., "protein + adjuvant" immunization mode). In addition, since the main antigen epitopes targeted by current antibody drugs and cell immunotherapy immune targets such as chimeric antigen receptors are located outside the target cells, in order to prepare monoclonal antibodies that specifically recognize the extracellular epitopes of membrane proteins, it is usually necessary to artificially construct and synthesize in vitro the extracellular regions of natural membrane proteins or even more precise protein epitopes to replace the full-length proteins for immunizing mice, which not only further increases costs but also reduces the immediate availability of some proteins whose extracellular sequences have not yet been measured.
[0004] In summary, given the cost and efficiency issues faced by protein immunization methods in hybridoma immunization, there is an urgent need to design a more efficient and simple mouse immunization method. Summary of the invention
[0005] In response to the above problems, the present invention provides the use of BALB / c-3T3 fibroblasts that overexpress specific proteins in hybridoma preparation, mainly to solve the problem that the current preparation of hybridoma cells relies too much on the "protein + adjuvant" immunization model and has low preparation efficiency.
[0006] In order to solve the above problems, the present invention adopts the following technical solutions:
[0007] The first aspect of the present invention provides the use of fibroblasts BALB / c-3T3 in the preparation of hybridoma cell lines (equivalent to hybridomas). Specific fibroblasts BALB / c-3T3 can be used to immunize specific objects, and then hybridomas can be obtained, and further hybridoma cell lines can be obtained. This method is simpler than conventional methods, does not require any adjuvants, and can maintain a high positive rate.
[0008] The specific requirements for some conditions in this application are as follows, and any section and any feature in each section can be selected independently:
[0009] Wherein, the fibroblast BALB / c-3T3 overexpresses the target protein, and the target protein is the antigen molecule that the target antibody secreted by the obtained hybridoma cell line can target and bind. Further, the fibroblast BALB / c-3T3 is derived from mice; further, the fibroblast BALB / c-3T3 is derived from BALB / c mice. The target protein is in the form of a membrane protein when overexpressed in the cell.
[0010] The aforementioned antigen molecules can be adjusted according to the type of hybridoma to be prepared, and the corresponding antigen molecules can be adjusted according to the target antibody secreted by the hybridoma cell line; the antigen molecules are at least GPRC5D molecules, and can also be other membrane protein molecules that can serve as potential immune targets. The specific means of making fibroblasts BALB / c-3T3 overexpress the target protein can adopt existing technologies.
[0011] Among them, regarding the aforementioned fibroblast BALB / c-3T3 overexpressing the target protein, regarding its transformation means: the fibroblast BALB / c-3T3 overexpresses the target protein by lentivirus / retrovirus infection, transposon transduction, CRISPRa gene editing or any other method for the purpose of overexpressing genes; the more mature one is lentivirus transfection, because it is more stable and simple to operate, and the transfection rate is also high. Lentivirus transfection can be specifically carried out in the following steps: add the lentivirus transfer plasmid, packaging plasmid psPAX2 and envelope plasmid PMD.2G that overexpress the target protein gene sequence to a certain volume of DMEM culture medium, mix with PEI transfection-promoting solution diluent, transfect HEK-293T cells that have been starved with pure DMEM culture medium, and then supplement fetal bovine serum according to the configuration ratio of complete culture medium, and collect the upper concentrated virus by centrifugation.
[0012] Among them, BALB / c mice, which are the most commonly used in hybridoma preparation, can be selected as specific subjects.
[0013] In addition, if monoclonal antibodies are prepared based on this application, as long as the process involves the same applications as in this section, it should be regarded as an application of the scheme in this section.
[0014] The second aspect of the present invention provides the use of BALB / c-3T3 fibroblasts that overexpress a specific protein in hybridoma cell lines (equivalent to hybridomas); wherein the BALB / c-3T3 fibroblasts overexpress a target protein, and the target protein is an antigen molecule that can be targeted and bound by a target antibody secreted by the target hybridoma cell line to be obtained. The type of overexpressed target protein is adjusted according to the desired preparation of hybridomas that secrete different target antibodies. Cell lines that overexpress a specific protein generally use cell lines that normally express the protein as a control reference. When the expression level is higher than the normal level, it is overexpressed. Of course, if the partial expression level is equivalent to the normal expression, but still has a specific effect, it should be equivalent to the scope of the present invention.
[0015] The specific requirements for some conditions in this application are as follows, and any section and any feature in each section can be selected independently:
[0016] Among them, the fibroblast BALB / c-3T3 overexpressing a specific protein may not be used in combination with an adjuvant. When the fibroblast BALB / c-3T3 overexpressing a specific protein is used, it does not rely on an adjuvant and can be used alone to obtain a hybridoma. The "adjuvant" mentioned in this section is the adjuvant involved in the current conventional scheme, which is mainly an immune adjuvant (for details, please refer to the specific requirements of the existing conventional scheme, such as Freund's adjuvant, oil emulsion adjuvant, aluminum salt adjuvant, etc.), while this scheme eliminates the dependence on immune adjuvants, etc., and hybridomas can be prepared alone by using fibroblast BALB / c-3T3 overexpressing a specific protein. Fibroblast BALB / c-3T3 also replaces the antigen protein (generally a commercial protein agent) in the original conventional scheme. At present, it is only necessary to directly use fibroblast BALB / c-3T3 overexpressing a specific protein, and there is no need to prepare hybridomas by the "protein + adjuvant" method as before. Of course, although this clause involves "not in combination with an adjuvant", it does not mean that the present invention excludes this scheme, but only means that even without an adjuvant, it can achieve the same or even better effect as the conventional scheme. If in the process of preparing hybridomas, this scheme is used and an adjuvant is added, then even if an adjuvant is added, it should be regarded as the application of this scheme.
[0017] Wherein, the fibroblast BALB / c-3T3 is transfected by lentivirus to overexpress the target protein, and the fibroblast BALB / c-3T3 is derived from BALB / c mice. Further, the fibroblast BALB / c-3T3 is derived from BALB / c mice, and the target protein is in the form of a membrane protein when overexpressed in the cell; further, the antigen molecule is at least a GPRC5D molecule, and can also be other membrane protein molecules that can serve as potential immune targets.
[0018] In addition, if monoclonal antibodies are prepared based on this application, as long as the process involves the same applications as in this section, it should be regarded as an application of the scheme in this section.
[0019] The third aspect of the present invention provides a method for preparing a hybridoma, comprising the following steps:
[0020] S1. Obtain BALB / c-3T3 fibroblasts overexpressing the target protein;
[0021] S2. Inject BALB / c-3T3 fibroblasts overexpressing the target protein into BALB / c mice, and immunize the mice to obtain hybridomas.
[0022] The fibroblast BALB / c-3T3 overexpresses the target protein, and the target protein is the antigen molecule that can be targeted and bound by the target antibody secreted by the target hybridoma cell line to be obtained.
[0023] Among them, BALB / c-3T3 fibroblasts overexpressing the target protein were obtained: the lentiviral transfer plasmid, packaging plasmid and envelope plasmid overexpressing the target protein gene sequence were mixed and packaged into lentivirus, and the BALB / c-3T3 cells were infected with the lentivirus to obtain BALB / c-3T3 fibroblasts overexpressing the target protein.
[0024] When mice are immunized to obtain hybridomas: Step S3 may also be specifically included: selecting mice with serum antibody titers that meet the standards, performing booster immunization again, taking spleen cells and fusing them with myeloma cells to culture hybridoma cell lines, and by booster immunization, spleen B cells can reach the best state. Furthermore, step S3 specifically includes: taking spleen cells and grinding them to obtain a cell suspension, adding preheated PEG1450 and letting it stand at 37±0.5°C, adding preheated 1640 to terminate the action of PEG1450, centrifuging, resuspending in the configured HAT culture medium, and adding to the well plate with feeder cells. Some of the reaction conditions introduced therein are not limited to this, and other conditions with similar effects should be within the scope of the present invention.
[0025] In the present disclosure, the target hybridoma can be directly obtained by immunizing BALB / c-3T3 fibroblasts that overexpress a specific protein, with a high positive rate, and can be used alone to prepare hybridoma cell lines without adjuvants, and the type of overexpressed target protein can be directly changed to prepare different target hybridomas. In addition, there is no need to purchase commercial proteins, which reduces the cost of hybridoma preparation. At present, the comprehensive cost can be reduced to half of the original cost or even lower. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 Double verification of antigen overexpression level by flow cytometry and qPCR;
[0027] Figure 2 Mouse serum antibody titer detection - cell-based ELISA results;
[0028] Figure 3 For mouse serum antibody titer detection - protein-based ELISA test;
[0029] Figure 4 The ELISA was used to detect the antibody level in the supernatant on the 12th day after fusion, and the positive well rate could reach more than 90%;
[0030] Figure 5 Retest the best positive well obtained in the initial screening three days after rehydration;
[0031] Figure 6 The fusion results are shown after BALB / c mice were immunized with the mouse fibroblast cell line NIH-3T3 overexpressing the same protein (GPRC5D). DETAILED DESCRIPTION
[0032] The present invention is further described below in conjunction with specific research examples.
[0033] Experimental Materials
[0034] Main reagents and consumables: BALB / c-3T3 cell line (ATCC); HEK-293T cell (ATCC); sp2 / 0 cell line (ATCC); recombinant target antigen overexpression lentiviral plasmid (Jikai Biotechnology); psPAX2 plasmid (Wuhan Sian Medical Technology Co., Ltd.), pMD2.G plasmid (Wuhan Sian Medical Technology Co., Ltd.); puromycin (MDBio, Cat#P012-25mg); trypsin (Gibco, Cat#25200-056); 6 4-6 week old BALB / c-3T3 mice (Viton Lihua); high glucose DMEM culture medium (Gibco, Cat#C1995500BT); fetal bovine serum (Gibco, Cat#10099-141); RNA extraction kit (Kangwei Century, Cat#CW0581M), reverse transcription kit III RT SuperMix for qPCR (Vazyme, Cat#R333-01); qPCR kit ChamQ SYBR qPCR Master Mix (Vazyme, Cat#Q311-02 / 03); skimmed milk powder (BioFroxx, Cat#1172GR100); Tween 20 (Beyotime, Cat#ST825-100ml); PBS pH 7.4 buffer (Gibco, Cat#C0010500BT); HRP goat anti-mouse secondary antibody (Jackson, RRID:AB_2338513), TMB color development solution (Beyotime, Cat#P0209-100ml), TMB color development stop solution (Beyotime, Cat#P0215-100ml).
[0035] Main equipment: conventional PCR instrument (BIO-CENER); real-time fluorescence quantitative PCR instrument (BIO-RAD); flow cytometer (BD FACSVia); inverted fluorescence microscope (OLYMPUS IX71); spectrophotometer (Infinite F50).
[0036] Experimental methods
[0037] 1) The recombinant target antigen (such as GPRC5D molecule) overexpression lentiviral transfer plasmid, packaging plasmid psPAX2 and envelope plasmid PMD.2G are added to a certain volume of DMEM culture medium according to the system of 30μg:20μg:10μg (this system can be adjusted according to the size of the inserted sequence fragment of the transfer plasmid), mixed with PEI transfection-promoting solution diluent (the latter is slowly dripped into the former and allowed to stand at room temperature for 30 minutes), and HEK-293T cells that have been starved for 1 hour with pure DMEM culture medium are transfected. Fetal bovine serum is supplemented after 6 hours according to the configuration ratio of complete culture medium. After 72 hours, the supernatant is collected and added to an ultrafiltration tube for centrifugation (3500r4℃30min), the upper concentrated virus is collected and the titer is determined; BALB / c-3T3 cells are infected with lentivirus at MOI=10, and the medium is changed after 72 hours, and antigen-expressing positive cells are selected using appropriate concentrations of puromycin.
[0038] 2) The antigen-overexpressing BALB / c-3T3 cells were digested with trypsin, washed twice with PBS and resuspended, and the expression intensity of the gene expression indicator fluorescent gene zsGreen was detected by flow cytometry. Untreated BALB / c-3T3 cells were used as negative controls, and the screening was preferably performed until the fluorescence positivity rate reached more than 95%.
[0039] 3) BALB / c-3T3 cells overexpressing antigens were digested with trypsin, washed twice with PBS, and then centrifuged to obtain cell pellets, which were resuspended in Trizol and placed at room temperature for 5 minutes. RNA was extracted using an RNA extraction kit according to the supplier's instructions, and the RNA purity and concentration were detected using NanoDrop. According to the supplier's instructions, genomic DNA was removed from the RNA, RNA was reverse transcribed to obtain cDNA, and then fluorescent quantitative PCR was performed to compare the up-regulation fold of the target gene expression.
[0040] 4) Adjust the density of BALB / c-3T3 cells overexpressing antigens to 1E8 / ml, and inject the mice intraperitoneally at a volume of 100μl per BALB / c mouse. Immunize once every two to three weeks, for a total of 4 times. Seven days after the fourth immunization, use a capillary glass tube to collect blood from the retro-orbital venous plexus, and collect about 50-100μl of venous blood from each mouse. Place the collected blood sample at room temperature for 1.5 hours, wait for it to coagulate and shrink into a blood clot, and the upper serum can precipitate naturally. After 3000r, discard the precipitate and absorb the supernatant into a new EP tube after 10 minutes.
[0041] 5) Cell ELISA test: According to the previous gradient pre-experiment, the density of 96-well plate cell plating was determined, and the CHO-GPRC5D cells were adjusted to 8E5 / ml, 100μl per well, and placed in an incubator for overnight attachment. The next day, the 96-well plate culture medium was removed, washed once with PBS, and then fixed with 4% paraformaldehyde for 15 minutes. After fixation, it was washed once with PBS. Then 200μl of PBSM (PBS+5% milk powder) was added to each well and placed in a 37-degree Celsius constant temperature incubator for 2h, and the plate was washed 4 times, each time for more than 3min. Then, mouse serum dilutions diluted according to different titers were added, and the plate was washed 5 times after incubation at room temperature for 1h, each time for more than 3min. HRP sheep anti-mouse antibody was diluted with PBSM at 1:5000, 100μl was added to each well, incubated at room temperature for 30min, and the plate was washed 5 times, each time for more than 3min. Add 100μl of TMB colorimetric solution to each well, incubate at room temperature for 15-20 minutes, and observe the color development. Then add TMB colorimetric stop solution and wait for the blue color to turn yellow before testing the OD value corresponding to the 450nm filter. Use the blank well as zero, and the dilution factor corresponding to the positive well / negative well (i.e., unimmunized mice) ratio > 3 is the titer of the mouse serum.
[0042] 6) Protein ELISA test: adjust the antigen protein concentration to 0.5 μg / ml, add 100 μl per well to the high affinity ELISA plate, and place in a 4°C refrigerator overnight. Omit the paraformaldehyde fixation step, and the remaining steps are the same as the cell ELISA experiment. Select the two mouse sera with the highest titer in the cell ELISA and the negative mouse sera collected in advance without immunization for the next step.
[0043] 7) Hybridoma fusion: Select mice with the highest serum antibody titer, and perform booster immunization again three days in advance. On the day of fusion, the mice were killed, and the spleen was removed by sterile operation. The spleen cells were obtained by grinding and resuspended in 10ml 1640. 200μl was taken for red cleavage and counted to estimate the total number of spleen cells. Sp2 / 0 and spleen cells were mixed in a 1:1 ratio, washed twice with 1640 medium and centrifuged. The tube wall was tapped to loosen the cells. Preheated PEG1450 was added at 37 degrees Celsius and allowed to stand for 1 minute. Preheated 1640 was added to terminate the effect of PEG1450. The tube was centrifuged at 1500r for 6 minutes, resuspended in the prepared HAT medium, and the feeder cells that were pre-plated were added (the feeder cells were peritoneal macrophages of spare BALB / c mice taken the day before the fusion, and the plating density was 2×10 4 96-well plates (cells / well), 2-3 drops per well, replenish the solution once on the third day after fusion, and after 10-14 days, when the density of hybridoma cells increases to one-tenth of the bottom of the well, aspirate the supernatant for ELISA detection, and retest the positive wells three days after changing the solution.
[0044] Results Analysis
[0045] 1) Double verification of antigen overexpression level by flow cytometry and qPCR, such as Figure 1 As shown in the figure, it was confirmed that the antigen (in this case, the human GPRC5D molecule) was successfully overexpressed after lentiviral transfection compared with the non-overexpressed cell line BALB / c-3T3, and compared with the human myeloma cell lines MM.1S, RPMI 8226 and human chronic myeloid leukemia cell line K562 that normally express the human GPRC5D molecule, the GPRC5D level of the overexpressed BALB / c-3T3 showed a significant increase.
[0046] 2) Mouse serum antibody titer detection - cell-based ELISA results, as shown in the following table and Figure 2 As shown in the figure, the blank control wells were analyzed as positive if the value was 3 times greater than the negative control value after zero adjustment. The antibody titers of the five mice were all greater than the maximum dilution factor of 2187000, indicating the high efficiency of this method.
[0047]
[0048] 3) Mouse serum antibody titer detection - protein-based ELISA test, such as Figure 3 As shown in , the antibody titers of immune mouse 2 (Yang 2) and immune mouse 1 (Yang 1) are at least 729,000, meeting the requirements for fusion.
[0049] 4) Specific antibody detection of hybridoma supernatant 12 days after fusion, such as Figure 4 As shown in the figure, taking plates 9 and 10 as examples (since it is difficult to distinguish the color depth with the naked eye after termination, this is the result before termination), the antibody level in the supernatant was detected by ELISA on the 12th day after fusion, and the positive hole rate could reach more than 90%.
[0050] 5) Retest the best positive well obtained in the initial screening three days after rehydration. Figure 5 As shown in , re-examination suggested that a few positive wells had lost their advantage, but the positive rate was still over 90%, and the probability of obtaining a positive monoclonal clone subsequently was very high.
[0051] 6) The fusion results after previously using the NIH mouse fibroblast cell line NIH-3T3 to overexpress the same protein and immunizing BALB / c mice in the same way as in this project, such as Figure 6 As shown in Figure 5, it can be seen that the immune effect of NIH-3T3 (plate 5, plate 8) overexpressing the same protein is not as good as that of BALB / c-3T3 ( Figure 4 and Figure 5), the positive rate and positive degree were significantly lower than the latter. This shows that not all fibroblast cell lines have the expected effect. For example, the fusion of NIH mouse fibroblast cell line NIH-3T3 overexpressing the same protein to immunize BALB / c mice has a poor effect and cannot effectively prepare hybridomas, while BALB / c-3T3 cells can achieve the expected effect.
[0052] Based on the above, after the BALB / c-3T3 cells overexpressing the target antigen protein in the present invention enter the mouse body, the BALB / c mice have a high homology to the BALB / c-3T3 cells, and the mouse immune system will not produce an immune response to most of the antigens on the membrane, but mainly recognize the extracellular segment of the target protein artificially overexpressed on the cell surface, so fewer mixed antibodies are produced, and it is easier to obtain monoclonal antibodies against the extracellular segment of the target protein. Compared with conventional protein immunization methods, this method has a lower cost, mainly from the cost of obtaining cell lines and constructing overexpression lentiviral plasmids. While not affecting the immune timeliness, it greatly reduces the cost required for the experiment, and the use of BALB / c-3T3 vectors can arbitrarily adjust the type of overexpressed protein to construct overexpression cell lines according to different targets, and obtain hybridomas specific to different targets.
[0053] It is clear to those skilled in the art that various modifications to the above embodiments can be made without departing from the overall spirit and concept of the present invention, and reference can be made to the prior art for any details not detailed herein. All of them fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims attached to the present invention.
Claims
1. Application of BALB / c-3T3 fibroblasts in the preparation of hybridoma cell lines.
2. The use according to claim 1; wherein The fibroblast BALB / c-3T3 overexpresses the target protein, and the target protein is the antigen molecule that can be targeted and bound by the target antibody secreted by the target hybridoma cell line to be obtained.
3. The use according to claim 2; wherein: The fibroblast BALB / c-3T3 is overexpressed with a target protein by at least one of lentivirus / retrovirus infection, transposon transduction, and CRISPRa gene editing.
4. The use according to claim 3; wherein: The fibroblast BALB / c-3T3 is derived from BALB / c mice; the antigen molecule is at least a GPRC5D molecule.
5. The use according to claim 2; wherein: The fibroblast BALB / c-3T3 is derived from mice; and the target protein is a membrane protein.
6. Application of BALB / c-3T3 fibroblasts overexpressing specific proteins in hybridoma cell lines; The fibroblast BALB / c-3T3 overexpresses the target protein, and the target protein is the antigen molecule that can be targeted and bound by the target antibody secreted by the target hybridoma cell line to be obtained.
7. The use according to claim 6; wherein: The BALB / c-3T3 fibroblasts overexpressing a specific protein are not combined with an adjuvant, and the BALB / c-3T3 fibroblasts are transfected by a lentivirus to overexpress the target protein, and the BALB / c-3T3 fibroblasts are derived from BALB / c mice.
8. A method for preparing a hybridoma, characterized in that: The steps include: S1. Obtain BALB / c-3T3 fibroblasts overexpressing the target protein; S2. Inject BALB / c-3T3 fibroblasts overexpressing the target protein into BALB / c mice, and immunize the mice to obtain hybridomas.
9. The method for preparing a hybridoma according to claim 8, characterized in that: The method for obtaining the overexpression fibroblast BALB / c-3T3 comprises at least: mixing and packaging a lentiviral transfer plasmid, a packaging plasmid and an envelope plasmid that overexpress a target protein gene sequence into a lentivirus, and infecting BALB / c-3T3 cells with the lentivirus; immunizing mice to obtain hybridomas, including step S3: selecting mice with serum antibody titers that meet the standard, performing booster immunization again, and taking spleen cells to fuse with myeloma cells to culture hybridoma cell lines.
10. The method for preparing a hybridoma according to claim 9, characterized in that: Step S3 specifically includes: taking spleen cells and grinding them to obtain a cell suspension, adding preheated PEG1450 and letting it stand at 37±0.5°C, adding preheated 1640 to terminate the effect of PEG1450, centrifuging, resuspending in the prepared HAT culture medium, and adding to the well plate with feeder cells.